Despite recent advances in understanding of mammalian embryonic hematopoiesis, site of origin and developmental potential of embryonic hematopoietic stem cells (HSC) remain unclear. Furthermore, recent observation that neuronal stem cells could have a much broader developmental capacity than anticipated, calls for reassessment of the full developmental potential of other tissue-specific stem cell types (e.g. HSC, mesenchymal, hepatic, epidermal stem cells etc.). New in utero transplantation assay termed blastocyst engraftment assay (BEA) will be used to further study embryonic hematopoiesis. BEA is based on microinjection of purified HSC into mouse preimplantation embryos (blastocysts), similar to ES cell technology. This new assay does not interfere with embryo development and results in engraftment of donor HSC in embryonic and adult hematopoietic tissues of recipients. The advantage of BEA is that the trafficking of donor HSC takes place during normal embryonic development, and secondly, the engraftment of donor HSC occurs through competitive repopulation of newly forming cell niches. The overall goal of the proposed project is to (a) study homing and engraftment of microinjected transgenic fetal and adult HSC during mouse embryo development in vivo and in vitro, and (b) comparatively analyze the potential of mouse fetal and adult HSC for development into non-hematopoietic cells/tissues. Towards this goal, the first part of the proposal focuses on the extensive in vivo analysis of the fate of transgenic fetal and adult HSC (isolated from ROSAbeta-geo26 and GFP transgenic mice) after microinjection into embryos of wild type and GATA-2 and SCL knockout mice. Engraftment of donor HSC in embryonic hematopoietic tissues will be assessed by analysis of embryos, tissues and cells using histochemical X-gal staining, fluorescence microscopy, histological and molecular techniques and in vitro hematopoietic colony forming assays (CFU-C). To complement in vivo studies, the second part deals with in vitro analysis of the fate of microinjected transgenic HSC. Culture of whole mouse embryos (reaching developmental stage of day 10 embryos in vivo) will be used as an experimental system for real time tracking of GFP-expressing transgenic HSC within the same embryos over the course of culture. The third part encompasses in vivo analysis of the potential of fetal and adult mouse HSC to differentiate into non-hematopoietic cells during mouse embryo development. The outcome of proposed studies could have important impact on the fields of embryonic hematopoiesis and clinical in utero and adult transplantation of HSC, .and perhaps on the development of new therapeutic approaches using autologous HSC to treat disorders or injuries affecting non-hematopoietic, tissues where the specific stem cells are not readily available for isolation.